Fructose-bisphosphate aldolase A (ALDOA) is an ancient, highly expressed gene that has acquired three distinct cellular activities. The best understood activity is catalyzing a key step in aerobic glycolysis. ALDOA also has protein binding activities independent of its catalytic activity (?moonlighting?), that include binding to cytoskeletal proteins, which use an ?E?E? motif that fits into a ?moonlighting pocket? of ALDO. Binding to the cytoskeleton actin holds ALDO in an inactive form until it is needed, when it is released by an increase in the levels of its substrate fructose-1, 6-bisphosphate and/or by growth-factor-induced PI-3-kinase activity. Another moonlighting function of ALDOA is its presence in the nucleus of cancer cells, where it is associated with increased proliferation. One possibility is that nuclear ALDOA binds to the E?E motif on EID-1 in the nucleus to inhibit HIF-1's transcriptional co-activator, p300. ALDOA levels are increased in many cancers, particularly pancreatic cancer, where it has been linked to poor patient survival and an increase in metastasis. Pancreatic cancer cell has high levels of anaerobic glycolysis that is further increased by the hypoxia inducible transcription factor-1 (HIF-1). HIF-1 induces ALDOA and other glycolytic enzymes that in turn maintain high HIF-1 activity through an AMPK/p300-dependent feed-forward loop. HIF-1 activity leads to VEGF release and angiogenesis, and the induction of other cancer cell survival mechanisms. Increased glycolysis provides hypoxic cancer cells with an increased supply of energy (ATP) and essential metabolites for biomass synthesis. Elevated ALDOA is also associated with low E-cadherin, a component of cancer cell tight junctions (TJ) necessary for cell-cell interactions, giving a more mesenchymal phenotype and increased tumor metastasis. This most likely is due to binding of ALDOA to the E?E motif of cytoskeleton actin causing its polymerization and disassembly, or other inactivation of the TJ with loss of E-cadherin. All this makes ALDOA an exceptional druggable anti-cancer target. Our X-ray crystallography studies have identified a new role for the C-terminal domain of ALDO in its catalytic activity, as well as a reactive Cys289 residue that allows allosteric regulation of catalytic activity. We have identified a novel lead probe allosteric inhibitor of ALDOA that forms a complex with Cys289 inhibiting glycolysis, HIF-1 activity and the proliferation of cancer cells, and in vivo inhibits glycolysis and tumor growth in a tumor xenograft model. Using this biologically stable allosteric inhibitor as a lead, one of our goals is to make more potent inhibitors that bind to the allosteric site Cys289, as well as high affinity bifunctional inhibitors that simultaneously engage the active site and moonlighting pocket. We will use X-ray crystallography to help us design inhibitors that modulate nuclear regulators of HIF-1 activity; and not least to design direct inhibitors of ALDOA catalytic activity. We will use these compounds as pharmacological probes to inhibit the various activities of ALDOA, and as potential leads for new therapies for the treatment of pancreatic, and other cancers.